Double-stranded cDNA fragments were synthesized from hepatitis A virus (HAV) RNA and inserted into the Pst I site of pBR322. The identity of cloned cDNA was established by demonstating its hydridization to RNA from HAV-infected tissue culture cells but not to RNA from uninfected cells. Genomic length RNA of approximately 7500 nucleotides was the predominant species that hybridized with the HAV cloned HAV cDNA. Restriction endonuclease digestion and hybridization between subgenomic fragments yielded a map of overlapping cloned cDNAs which included 99% of the viral genome. Cloned cDNA from near the 5' terminus of the genome was used to synthesize and clone cDNA by primer extension so that molecular clones could be obtained that contained the 5' terminus of the genome. Cloned cDNA was used as a probe for detecting HAV RNA in tissue culture and fecal specimens by hybridization.